Bottom Line:
This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness.As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO.The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962.

Affiliation: Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.

Figure 1: WashU and CDC RT-PCR design comparison. WashU and CDC RT-PCR primers and probe locations are illustrated within the VP1 gene of the 2014 outbreak EV-D68 St. Louis (GenBank accession no. KM881710.2) reference genome. The dark green and dark blue areas, as well as the light yellow bounding box, indicate the regions of the genome targeted by the respective assays and their associated PCR product lengths. Arrows indicate direction of priming for the left (L) and right (R) primers. The yellow squares labeled F (fluorescent reporter) and the green squares labeled Q (quencher) show the relative orientation of the fluorophores on the probes.

Mentions:
Based on this procedure, we selected an RT-PCR set consisting of two primers and a single probe with complete sequence identity to the 2014 outbreak virus (WashU design 1). To broaden the detection of EV-D68 viruses, we made modifications based on SNP frequencies that included the addition of degenerate bases and a second reverse primer (WashU design 2). Both designs are shown in Table 1 and Fig. 1.

Figure 1: WashU and CDC RT-PCR design comparison. WashU and CDC RT-PCR primers and probe locations are illustrated within the VP1 gene of the 2014 outbreak EV-D68 St. Louis (GenBank accession no. KM881710.2) reference genome. The dark green and dark blue areas, as well as the light yellow bounding box, indicate the regions of the genome targeted by the respective assays and their associated PCR product lengths. Arrows indicate direction of priming for the left (L) and right (R) primers. The yellow squares labeled F (fluorescent reporter) and the green squares labeled Q (quencher) show the relative orientation of the fluorophores on the probes.

Mentions:
Based on this procedure, we selected an RT-PCR set consisting of two primers and a single probe with complete sequence identity to the 2014 outbreak virus (WashU design 1). To broaden the detection of EV-D68 viruses, we made modifications based on SNP frequencies that included the addition of degenerate bases and a second reverse primer (WashU design 2). Both designs are shown in Table 1 and Fig. 1.

Bottom Line:
This assay was developed in response to the unprecedented 2014 nationwide EV-D68 outbreak in the United States associated with severe respiratory illness.As part of our evaluation of the outbreak, we sequenced and published the genome sequence of the EV-D68 virus circulating in St. Louis, MO.The assay did not detect any other enteroviruses or rhinoviruses tested and did detect divergent strains of EV-D68, including the first EV-D68 strain (Fermon) identified in California in 1962.

Affiliation:
Department of Pediatrics, Washington University School of Medicine, St. Louis, Missouri, USA McDonnell Genome Institute, Washington University School of Medicine, St. Louis, Missouri, USA.